Derivatives of korormicin useful as antibiotics

ABSTRACT

or a salt or stereoisomer thereof; wherein R1-R3 and R5-R10 are independently selected from the group consisting of H, alkyl group, substituted alkyl group, halogen, OH, NH2 and SH; R4 is H, alkyl group or substituted alkyl group; X1-X2 are independently selected from the group consisting of ═O, ═S, NH, H, alkyl, halogen, OH, SH and NH2; W is a saturated acyclic hydrocarbon chain of 1 to 15 carbon atoms; and Z is a neutral or positively charged organic group. The compounds are useful in treating bacterial diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/500,006, filed Jan. 27, 2017, which is the U.S. national phase ofInternational Application No. PCT/CA2016/050470, filed Apr. 22, 2016,which claims benefit of U.S. Provisional Application No. 62/151,902,filed Apr. 23, 2015, all of which are expressly incorporated byreference in their entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to antibiotic compounds and therapeuticuses thereof.

Description of the Related Art

A global crisis regarding treatment and control of bacterial infectionsis well recognized. Medical concerns include a lack of sufficientvariety of antibiotics to address new infections emerging in the worldand resistance to conventional antibiotic treatment that has beenobserved in adapting bacteria. New antibiotics to address the situationhave not been created. In the mid-1980's 16 new antibiotics wereapproved by the FDA. In 2008/09, this had decreased to just one(Infectious Diseases Society of America). During the same approximatetime period, resistance to antibiotics had climbed from an incidence of˜5% to 30-60%. This has prompted the World Health Organization to statein 2009 that the “rapid development of anti-microbial resistance is oneof the three greatest threats to human health.”

Part of the problem in identifying new drugs to address this crisis isthat industry has depleted itself of targets to control bacteria. Thetargets that have been conventionally used in the past and are commonfor all bacteria today include prokaryotic ribosomes, enzymes working onDNA & RNA, and the synthesis of cell walls, etc. Virtually no newmechanistic targets have been identified.

The Na⁺-translocating NADH: ubiquinone oxidoreductase (Na⁺-NQR) inbacteria has been suggested as a mechanism to control bacterialreplication and viability. Three inhibitors of Na⁺-NQR have beenreported so far: denaturing Ag⁺ ions,(3R,4Z,6E)-N-[(5S)-5-Ethyl-5-methyl-2-oxo-2,5-dihydro-3-furanyl]-3-hydroxy-8-[(2 S,3R)-3-octyl-2-oxiranyl]-4,6-octadienamide(korormicin), and 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO). Ag⁺ ionshave obvious delivery problems and are non-specific as well. Bothkorormicin and HQNO have been shown to inhibit the sodium-dependentubiquinone reduction by Na⁺-NQR in a mutually exclusive manner and bothhave been suggested to possess antimicrobial activity (as discussed forexample, in Japanese Patent 3905604B2 published 18 Apr. 2007, JapanesePatent Application Publication 2000336088 published 5 Dec. 2000, andJapanese Patent Application Publication 2001010908 published 16 Jan.2001). However, none of these compounds have been developed as aneffective antibiotic treatment for animals, for example mammals, or moreparticularly humans.

Accordingly, there is a continuing need for alternative antibioticcompounds.

SUMMARY OF THE INVENTION

In an aspect there is provided, an antibiotic compound of formula (III):

or a salt or stereoisomer thereof;

wherein R¹-R³ and R⁵-R¹⁰ are independently selected from H, alkyl group,substituted alkyl group, halogen, OH, NH₂, or SH;

R⁴ is H, alkyl group or substituted alkyl group;

X¹-X² are independently selected from O, S, NH, H, alkyl, halogen, OH,SH, or NH₂;

W is a saturated acyclic hydrocarbon chain having 1 to 15 carbon atomsconsisting essentially of hydrogen and carbon atoms;

Z is CH₃ or any neutral or positively charged group.

In another aspect there is provided, an antibiotic compound of formula(I):

or a salt or stereoisomer thereof;

wherein R¹-R³, R⁵ and R⁶ are independently selected from H, alkyl group,substituted alkyl group, halogen, OH, NH₂, or SH;

R⁴ is H, alkyl group or substituted alkyl group;

X¹-X² are independently selected from O, S, NH, H, alkyl, halogen, OH,SH, or NH₂;

Y is an acyclic hydrocarbon chain having 2 to 20 carbon atoms or asubstituted acyclic hydrocarbon chain having 2 to 20 carbon atoms withthe proviso that Y does not include an oxygen atom;

Z is CH₃ or any neutral or positively charged group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an experimental design for assessment of an effect ofkorormicin on C. trachomatis infection of human cells.

FIG. 2 shows an experimental design for assessment of an effect of PEG-2on C. trachomatis infection of human cells.

FIG. 3 shows an experimental design for titration of P5 PEG-2 treatmentsshown in FIG. 2.

FIG. 4 shows a structural alignment comparison of korormicin and PEG-2.

FIG. 5 shows an effect of korormicin on C. trachomatis infection of HeLacells.

FIG. 6 shows a decline of intruding C. trachomatis after 5 consecutivetreatments with korormicin.

FIG. 7 shows a toxic effect of korormicin for primary Vascular SmoothMuscle cells.

FIG. 8 shows an absence of toxic effect of PEG-2 for primary VascularSmooth Muscle cells.

FIG. 9 shows an effect of PEG-2 on C. trachomatis infection of HeLacells.

FIG. 10 shows a quantification of the effect shown in FIG. 9.

FIG. 11 shows a decline of intruding C. trachomatis after 5 consecutivetreatments with PEG-2.

FIG. 12 shows a PEG-2 treatment (10 μM) of C. trachomatis infected HeLacells as a function of passage number.

FIG. 13 shows a PEG-2 treatment (15 μM) of C. trachomatis infected HeLacells as a function of passage number.

FIG. 14 shows a comparison of efficacy of PEG-2 versus korormicin inHeLa cells infected with C. trachomatis.

FIG. 15 shows chemical structures of homoserine lactones tested for HeLacell toxicity and anti-chlamydial efficacy.

FIG. 16 shows an effect of PEG-2 on initial C. trachomatis-inducedacidification of the cell cytoplasm.

FIG. 17 shows an effect of PEG-2 on C. trachomatis-induced acidificationof the cell cytoplasm at various time-points after infection (0, 2hours, and 24 hours).

FIG. 18 shows changes in cytoplasmic pH (A) and Na⁺ content (B) causedby the C. trachomatis infecting the HEK293 cell culture in high glucosemedium.

FIG. 19 shows that PEG-2 acts in a dose-dependent manner to reduceinclusion numbers during chlamydial infection, with inclusion numberscounted after 2^(nd), 3^(rd), 4^(th), and 5^(th) consecutive treatments.

FIG. 20 shows that PEG-2S is highly selective anti-Na⁺-NQR agent in thatgrowth yield of three Na⁺-NQR-negative species belonging to benign gutmicroflora in liquid medium was not effected at any tested concentrationof added PEG-2S.

FIG. 21 shows inhibition of Na⁺-NQR by PEG-2S as measured directly insub-bacterial V. cholerae vesicles: (A) PEG-2S-sensitive oxidation ofdNADH is absent in Na⁺-NQR-deficient membranes (trace (a)), whileNa⁺-NQR-containing membranes oxidize dNADH in the PEG-2S-sensitivemanner (trace (b); and (B) normalized Na⁺-NQR activity (100% correspondsto the activity in the absence of the inhibitor) plotted as a functionof [PEG-2S] for calculation of the IC₅₀ of PEG-2S inhibition.

FIG. 22 shows that PEG-2S disrupts chlamydial infection in HEK293 cellculture:

treatment with 1 and 2.5 μM PEG-2S prevents changes of cytoplasmic pH(A) and [Na⁺] (B) in HEK293 cells infected by C. trachomatis (plots areaverages of 8 independent experiments with standard deviation shown,P<0.003).

FIG. 23 shows examples of chemical structures of derivatives of PEG-2.

FIG. 24 shows that derivatives of PEG-2 provide an effective treatmentagainst C. trachomatis infecting HeLa cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Compounds, formulations, compositions, methods of treatment, and methodsand systems of drug discovery described herein relate to formulae I, II,III, or IV, with formula I encompassing formulae II, III, or W, andformula III encompassing formula IV:

where R¹-R³ and R⁵-R¹⁰ are independently selected from H, alkyl group,substituted alkyl group, halogen, OH, NH₂, or SH;

R⁴ is H, alkyl group or substituted alkyl group;

X¹-X² are independently selected from O, S, NH, H, alkyl, halogen, OH,SH, or NH₂;

Y is an acyclic hydrocarbon chain having 2 to 20 carbon atoms or asubstituted acyclic hydrocarbon chain having 2 to 20 carbon atoms;

W is an acyclic hydrocarbon chain having 1 to 15 carbon atoms or asubstituted acyclic hydrocarbon chain having 1 to 15 carbon atoms;

Z is CH₃ or any neutral or positively charged group.

Formulae I, II, III and IV have in common a furanone ring N-linked to anacyclic side chain. As is apparent from formulae I, II, III and W boththe furanone ring and the N-linked side chain can accommodate varioussubstituents.

In certain embodiments, components of formulae I, II, III or W may befurther defined. In one example, R¹-R¹⁰ are independently selected fromH or a C1-C5 alkyl group or a C1-C5 substituted alkyl group. In anotherexample, R¹-R¹⁰ are independently selected from H or a C1-C4 alkyl groupor a C1-C4 substituted alkyl group. In another example, R¹-R¹⁰ areindependently selected from H or a C1-C3 alkyl group or a C1-C3substituted alkyl group. In another example, R¹-R¹⁰ are independentlyselected from H or a C1-C2 alkyl group or a C1-C2 substituted alkylgroup. In another example, R⁴ is H. In another example, R¹-R³ areindependently selected from H or C1-C2 alkyl, R⁵-R¹⁰ are independentlyselected from H or C1-C3 alkyl group, and R⁴ is H.

In another example, X¹-X² are independently selected from O, S, H, C1-C3alkyl, halogen, OH, SH. In another example, X¹-X² are independentlyselected from O, S, H, halogen, OH, SH. In another example, X¹-X² areindependently selected from H, O, S, OH, or SH. In another example,X¹-X² are independently selected from H, O or OH. In another example, X¹is O and X² is OH. In another example, X¹ is O and X² is H.

In another example, Y is an acyclic hydrocarbon chain having 4 to 20carbon atoms or a substituted acyclic hydrocarbon chain having 4 to 20carbon atoms. In another example, Y is an acyclic hydrocarbon chainhaving 2 to 14 carbon atoms or a substituted acyclic hydrocarbon chainhaving 2 to 14 carbon atoms. In another example, Y is an acyclichydrocarbon chain having 2 to 12 carbon atoms or a substituted acyclichydrocarbon chain having 2 to 12 carbon atoms. In another example, Y isan acyclic hydrocarbon chain having 2 to 10 carbon atoms or asubstituted acyclic hydrocarbon chain having 2 to 10 carbon atoms. Inanother example, Y is an acyclic hydrocarbon chain having 2 to 8 carbonatoms or a substituted acyclic hydrocarbon chain having 2 to 8 carbonatoms. In another example, Y does not include an epoxide group. Inanother example, Y does not include a bromine group. In another example,Y does not include an oxygen atom. In another example, Y includes atleast one carbon-carbon double bond. In another example, Y consistsessentially of hydrogen and carbon atoms, encompassing variation thatdoes not materially alter toxicity and expressly not including anyepoxide group. In another example, Y consists only of hydrogen andcarbon atoms.

In another example, W is an acyclic hydrocarbon chain having 1 to 12carbon atoms or a substituted acyclic hydrocarbon chain having 1 to 12carbon atoms. In another example, W is an acyclic hydrocarbon chainhaving 1 to 10 carbon atoms or a substituted acyclic hydrocarbon chainhaving 1 to 10 carbon atoms. In another example, W is an acyclichydrocarbon chain having 1 to 8 carbon atoms or a substituted acyclichydrocarbon chain having 1 to 8 carbon atoms. In another example, W isan acyclic hydrocarbon chain having 1 to 6 carbon atoms or a substitutedacyclic hydrocarbon chain having 1 to 6 carbon atoms. In anotherexample, W is an acyclic hydrocarbon chain having 1 to 4 carbon atoms ora substituted acyclic hydrocarbon chain having 1 to 4 carbon atoms. Inanother example, W does not include an epoxide group. In anotherexample, W does not include a bromine group. In another example, W doesnot include an oxygen atom. In another example, W consists essentiallyof hydrogen and carbon atoms and is saturated, encompassing variationthat does not materially alter toxicity and expressly not including anyepoxide group. In another example, W consists only of hydrogen andcarbon atoms.

In another example, Z does not include an epoxide group. In anotherexample, Z does not include a bromine group. In another example, Z doesnot include an oxygen atom. In another example, Z is CH₃ or an organicgroup. In another example, Z is an organic group comprising a nitrogenatom. In another example, Z is an organic group comprising a phosphorusatom. In another example, Z is CH₃ or a positively charged organicgroup. In another example, Z is a halogen. In another example, Z is CH₃,a triphenylphosphine (3PhP) group, a guanidine group, an aminoperimidinegroup, an amiloride group or a halogen. In another example, themolecular weight of the compound is less than 2000 Daltons. In anotherexample, the molecular weight of the compound is less than 1000 Daltons.

In another example, a compound of formulae I, II, III or IV will have atleast two chiral centers, for example 5S and 3′R chiral centers shown inFIG. 4. In another example, a compound of formulae I, II, III or IV willhave only two chiral centers, for example 5S and 3′R chiral centersshown in FIG. 4.

In another example, a compound of formulae I, II, III or IV is providedwhere R¹-R³ are independently selected from H or a C1-C3 alkyl group andR⁵-R¹⁰ are independently selected from H or a C1-C5 alkyl group; R⁴ isH; X¹-X² are independently selected from O, S, H, alkyl, halogen, OH,SH; Y is an acyclic hydrocarbon chain having 2 to 20 carbon atoms or asubstituted acyclic hydrocarbon chain having 2 to 20 carbon atoms; W isan acyclic hydrocarbon chain having 1 to 15 carbon atoms or asubstituted acyclic hydrocarbon chain having 1 to 15 carbon atoms; Z isa neutral or positively charged group comprising a nitrogen atom, andoptionally the nitrogen atom forms a covalent bond with a carbon atom ofY or W.

In another example, a compound of formulae I, II, III or W is providedwhere R¹-R³ are independently selected from H or a C1-C3 alkyl group andR⁵-R¹⁰ are independently selected from H or CH₃; R⁴ is H; X¹-X² areindependently selected from O, S, H, OH, or SH; Y is an acyclichydrocarbon chain having 2 to 12 carbon atoms or a substituted acyclichydrocarbon chain having 2 to 12 carbon atoms; W is an acyclichydrocarbon chain having 1 to 8 carbon atoms or a substituted acyclichydrocarbon chain having 1 to 8 carbon atoms; Z is a neutral orpositively charged group comprising a nitrogen atom, and optionally thenitrogen atom forms a covalent bond with a carbon atom of Y or W.

In another example, a compound of formulae I, II, III or W is providedwhere R¹-R³ are independently selected from H or a C1-C3 alkyl group andR⁵-R¹⁰ are independently selected from H or a C1-C5 alkyl group; R⁴ isH; X¹-X² are independently selected from O, S, H, alkyl, halogen, OH,SH; Y is an acyclic hydrocarbon chain having 2 to 20 carbon atoms or asubstituted acyclic hydrocarbon chain having 2 to 20 carbon atoms; W isan acyclic hydrocarbon chain having 1 to 15 carbon atoms or asubstituted acyclic hydrocarbon chain having 1 to 15 carbon atoms; Z isa neutral or positively charged group comprising a phosphorus atom, andoptionally the phosphorus atom forms a covalent bond with a carbon atomof Y or W.

In another example, a compound of formulae I, II, III or W is providedwhere R^(l)-R³ are independently selected from H or a C1-C3 alkyl groupand R⁵-R¹⁰ are independently selected from H or CH₃; R⁴ is H; X¹-X² areindependently selected from O, S, H, OH, or SH; Y is an acyclichydrocarbon chain having 2 to 12 carbon atoms or a substituted acyclichydrocarbon chain having 2 to 12 carbon atoms; W is an acyclichydrocarbon chain having 1 to 8 carbon atoms or a substituted acyclichydrocarbon chain having 1 to 8 carbon atoms; Z is a neutral orpositively charged group comprising a phosphorus atom, and optionallythe phosphorus atom forms a covalent bond with a carbon atom of Y or W.

In another example, a compound of formulae I, II, III or W is providedwhere R¹-R³ are independently selected from H or a C1-C3 alkyl group andR⁵-R¹⁰ are independently selected from H or a C1-C5 alkyl group; R⁴ isH; X¹-X² are independently selected from O, S, H, alkyl, halogen, OH,SH; Y is an acyclic hydrocarbon chain having 2 to 20 carbon atoms or asubstituted acyclic hydrocarbon chain having 2 to 20 carbon atoms; W isan acyclic hydrocarbon chain having 1 to 15 carbon atoms or asubstituted acyclic hydrocarbon chain having 1 to 15 carbon atoms; Z ishalogen.

In another example, a compound of formulae I, II, III or W is providedwhere R¹-R³ are independently selected from H or a C1-C3 alkyl group andR⁵-R¹⁰ are independently selected from H or CH₃; R⁴ is H; X¹-X² areindependently selected from O, S, H, OH, or SH; Y is an acyclichydrocarbon chain having 2 to 12 carbon atoms or a substituted acyclichydrocarbon chain having 2 to 12 carbon atoms; W is an acyclichydrocarbon chain having 1 to 8 carbon atoms or a substituted acyclichydrocarbon chain having 1 to 8 carbon atoms; Z is halogen.

In another example, a compound of formulae I, II, III or W is providedwhere R¹-R³ are independently selected from H or a C1-C3 alkyl group andR⁵-R¹⁰ are independently selected from H or a C1-C5 alkyl group; R⁴ isH; X¹-X² are independently selected from O, S, H, alkyl, halogen, OH,SH; Y is an acyclic hydrocarbon chain having 2 to 20 carbon atoms or asubstituted acyclic hydrocarbon chain having 2 to 20 carbon atoms; W isan acyclic hydrocarbon chain having 1 to 15 carbon atoms or asubstituted acyclic hydrocarbon chain having 1 to 15 carbon atoms; Z isCH₃.

In another example, a compound of formulae I, II, III or W is providedwhere R¹-R³ are independently selected from H or a C1-C3 alkyl group andR⁵-R¹⁰ are independently selected from H or CH₃; R⁴ is H; X¹-X² areindependently selected from O, S, H, OH, or SH; Y is an acyclichydrocarbon chain having 2 to 12 carbon atoms or a substituted acyclichydrocarbon chain having 2 to 12 carbon atoms; W is an acyclichydrocarbon chain having 1 to 8 carbon atoms or a substituted acyclichydrocarbon chain having 1 to 8 carbon atoms; Z is CH₃.

Compounds described herein may be used to treat a subject or patientthat is a human or non-human animal. Treatment of mammals iscontemplated, including for example humans, primates, rodents, dogs,cats, cows, pigs, horses, or sheep. Contemplated treatment of birdsinclude, for example, chicken or turkey.

“Treatment” or “treating” refers to therapy, prevention and prophylaxisand particularly refers to the administration of medicine or theperformance of medical procedures with respect to a patient, for eitherprophylaxis (prevention) or to cure the infirmity or malady in theinstance where the patient is afflicted.

“Therapeutic agent” refers to an agent capable of having a desiredbiological effect on a host. Antibiotic agents are an example oftherapeutic agents that are generally known to be chemical in origin, asopposed to biological, typically having a small molecule structure witha molecular weight of less than 2000 Daltons.

The term “therapeutically effective amount” refers to that amount of anagent, modulator, drug or other molecule which is sufficient to effecttreatment when administered to a subject in need of such treatment. Thetherapeutically effective amount will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, which can readily be determined by one of ordinary skill inthe art.

The term “modulation”, when used in reference to a functional propertyor biological activity or process (e.g., enzyme activity or receptorbinding), refers to the capacity to either up regulate (e.g., activateor stimulate), down regulate (e.g., inhibit or suppress) or otherwisechange a quality of such property, activity or process.

Modulators include, for example, a polypeptide, nucleic acid,macromolecule, complex molecule, small molecule, compound, species orthe like (naturally-occurring or non-naturally-occurring), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal cells or tissues, that may be capable of causing modulation.

Modulators may be evaluated for potential activity as inhibitors oractivators (directly or indirectly) of a functional property, biologicalactivity or process, or combination of them, (e.g., agonist, partialantagonist, partial agonist, inverse agonist, antagonist, antimicrobialagents, inhibitors of microbial infection or proliferation, and thelike) by inclusion in assays. In such assays, many modulators may bescreened at one time. The activity of a modulator may be known, unknownor partially known.

Dosage ranges for the administration of antibiotic compounds are readilydetermined by the skilled person through routine testing. The dosageshould not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any counterindications.

The dose, schedule of doses and route of administration may be varied,whether oral, nasal, vaginal, rectal, extraocular, intramuscular,intracutaneous, subcutaneous, or intravenous, and the like.

Antibiotic compounds described herein can be used therapeutically incombination with a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable refers to those ingredients, compositions and dosages thereofwithin the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable carriers are known to those skilled in the art. These mosttypically would be standard carriers for administration of compositionsto humans, including solutions such as sterile water, saline, andbuffered solutions at physiological pH.

Pharmaceutically acceptable carriers include liquid carriers, solidcarriers, or both. Liquid carriers include, but are not limited to,water, saline, physiologically acceptable buffers, aqueous suspensions,oil emulsions, water in oil emulsions, water-in-oil-in-water emulsions,site-specific emulsions, long-residence emulsions, sticky-emulsions,microemulsions and nanoemulsions. Examples of aqueous carriers includewater, saline and physiologically acceptable buffers. Examples ofnon-aqueous carriers include a mineral oil or a neutral oil including,but not limited to, a diglyceride, a triglyceride, a phospholipid, alipid, an oil and mixtures thereof. Solid carriers are biologicalcarriers, chemical carriers, or both and include, for example,particles, microparticles, nanoparticles, microspheres, nanospheres,minipumps, bacterial cell wall extracts, and biodegradable ornon-biodegradable natural or synthetic polymers that allow for sustainedrelease of the composition

Molecules intended for pharmaceutical delivery may be formulated in apharmaceutical composition. Pharmaceutical compositions may includeacceptable carriers, thickeners, diluents, buffers, preservatives,surface active agents and the like in addition to the molecule ofchoice. Pharmaceutical compositions may also include one or more activeingredients having biological activity such as antimicrobial agents,anti-inflammatory agents, anesthetics, allergy relief agents, painrelief agents and the like.

A composition may be administered in a number of ways depending onwhether local or systemic treatment is desired, and on the area to betreated. Administration may be topically (including ophthalmically,vaginally, rectally, intranasally), orally, by inhalation, orparenterally, for example by intravenous drip, subcutaneous,intraperitoneal or intramuscular injection. The compositions may beadministered according to standard procedures used by those skilled inthe art.

An effective dose or amount, and any possible effects on the timing ofadministration of the formulation, may need to be identified for anyparticular compound described herein. This may be accomplished byroutine experiment, using one or more groups of animals (for exampleusing at least 5 animals per group), or in human trials if appropriate.The effectiveness of any compound and method of treatment or preventionmay be assessed by administering the compound and assessing the effectof the administration by measuring one or more indices associated withthe disease or condition of interest, and comparing the post-treatmentvalues of these indices to the values of the same indices prior totreatment.

The precise time of administration and amount of any particular compoundthat will yield the most effective treatment in a given patient maydepend upon the activity, pharmacokinetics, and bioavailability of aparticular compound, physiological condition of the patient (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage and type of medication), route ofadministration, and the like.

While a subject is being treated, the health of the subject may bemonitored by measuring one or more of the relevant indices atpredetermined times during a 24-hour period. Treatment, includingsupplement, amounts, times of administration and formulation, may beoptimized according to the results of such monitoring. The subject maybe periodically reevaluated to determine the extent of improvement bymeasuring the same parameters, the first such reevaluation typicallyoccurring at the end of four weeks from the onset of therapy, andsubsequent reevaluations occurring every four to eight weeks duringtherapy and then every three months thereafter. Therapy may continue forseveral months or even years, with a minimum of two weeks being atypical length of therapy for humans. Adjustments to the amount(s) ofagent administered and possibly to the time of administration may bemade based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The combined use of several compounds described herein, or alternativelyother antibiotic agents, may reduce the required dosage for anyindividual component because the onset and duration of effect of thedifferent components may be complimentary. In such combined therapy, thedifferent active agents may be delivered together or separately, andsimultaneously or at different times within the day.

Toxicity and therapeutic efficacy of subject compounds may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 and the ED50. Compositions thatexhibit large therapeutic indices are advantageous. Although compoundsthat exhibit toxic side effects may be used, care should be taken todesign dosage ranges, formulations, or delivery systems that target thecompounds to the desired site in order to reduce side effects.

The data obtained from the cell culture assays and non-human animalstudies may be used in formulating a range of dosage for use in humans.The dosage of any supplement, or alternatively of any componentstherein, can lie within a range of circulating concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For agents described herein, thetherapeutically effective dose may be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationmay be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

Compounds described herein may be used for antibiotic treatment of asubject or patient that is a human or non-human animal. The antibiotictreatment can be useful in inhibiting growth of various bacteria andmodulating bacterial infections. Without wishing to be bound by theory,the antibiotic treatment may include an interaction with anNa⁺-translocating NADH: ubiquinone oxidoreductase (Na⁺-NQR) protein orpeptide and/or may include modulation of Na⁺-NQR activity.

Without wising to be bound by theory, the present inventors havedeveloped a model for explaining how the Na⁺ cycle and sodium motivepumps may play a role in the course of bacterial infection.Specifically, the model suggests targeting the Na⁺-translocating NADH:

ubiquinone oxidoreductase (Na⁺-NQR) in bacteria as a mechanism tocontrol bacterial replication and viability. Na⁺-NQR is an unusualrespiratory enzyme that functions as a primary sodium pump utilizing theenergy of oxidation of NADH by quinone to expel Na⁺ ions from thecytoplasm. It thus creates a sodium motive force that can be directlyused for metabolic work, including import of amino acids and operationof multidrug resistance pumps. Importantly, Na⁺-NQR is present in manypathogenic bacteria, but it is absent in mitochondria of human cells aswell as in the species belonging to normal gastroenteric microflora.Thus, specific targeting of this enzyme may result in less non-specificside effects in human cells and normal gastoenteric microflora thantraditionally prescribed antibiotics.

The mechanistic pathway and the involvement of Na⁺-NQR in bacterialmetabolism can occur in several steps. The consumption of any availableintracellular ATP by intruding bacteria results in a slowing of Na⁺ pumpwhich can decelerate the removal of Na⁺ from the infected cell.Stimulation of glycolysis by the invading bacteria can accentuate theNa⁺ overload indirectly by causing an acidification of the cytoplasmwhich ultimately activates the Na⁺/H⁺ exchanger (NHE1) to import moreNa⁺ into the cell in exchange for the removal of intracellular H⁺. Whenintracellular pools of ATP and glucose are exhausted, the pathogenicbacteria can switch to amino acid catabolism, which efficiently raisesthe pH of the cytoplasm. Intracellular concentration of Na⁺ ions remainselevated due to the depressed activity of the Na⁺ pump. Na⁺-NQR is anunusual respiratory enzyme that functions as a primary sodium pumputilizing the energy of oxidation of NADH by quinone to expel Na⁺ ionsfrom the cytoplasm. It thus creates a sodium motive force that can bedirectly used for metabolic work, including import of amino acids andoperation of multidrug resistant pumps. Under these conditions, thepathogenic bacteria rely on the Na⁺-NQR to energize the uptake of aminoacids, which can occurs via several pathways including Na⁺-amino acidsymports. Inhibition of Na⁺-NQR should, therefore, under theseconditions result in no ability to provide energy to support amino aciduptake over to re-establish normal ionic homeostasis.

Na⁺-NQR, is the major respiratory Na⁺ pump in aerobic pathogens.Phylogenetic analysis shows that variants of full Na⁺-cycle as well assole primary Na⁺ pumps are overrepresented in pathogenic species,possibly due to the difficulties for the intruding pathogens to maintainsufficiently high proton-motive force (PMF) in hostile microenvironmentsof a colonized macro-organism. Widespread distribution of Na⁺-NQR andits implication in the regulation of virulence traits make this enzymean attractive candidate for the development of novel antibiotics,especially after recent long-awaited breakthrough in studies of Na⁺-NQRstructure (Steuber et al. Structure of the V. cholerae Na⁺-pumpingNADH:quinone oxidoreductase. Nature. 2014; 516:62-67).

Na⁺-NQR is found in a number of pathogenic bacteria. The potential forit to affect the course of a wide range of pathogenic bacteria is,therefore, large. Potentially, any Na⁺-NQR-containing pathogenicbacterium could be sensitive to drugs targeting Na⁺-NQR. An incompleteand illustrative list of the many species that contain Na⁺-NQR and thediseases with which these bacteria are associated is shown in Table 1.

TABLE 1 Na⁺-NQR-containing bacteria and the diseases associated withthese infections. ORGANISM Gram DISEASE(S) CAUSED Vibrio cholerae (−)(cholera) Vibrio parahaemolyticus (−) (acute gastroenteritis) Vibriovulnificus (−) (ulcers, GE infections) Vibrio gastroenteritis (−) (acutegastroenteritis) Vibrio damsela (−) (wound infection, septicemia) Vibriofluvialis (−) (foodborne diarrhea) Vibrio furnissii (−) (foodbornediarrhea) Vibrio harveyi (−) (foodborne diarrhea) Vibrio hollisae (−)(foodborne diarrhea) Vibrio costicola (−) (foodborne diarrhea) Vibriomimicus (−) (foodborne diarrhea) Vibrio cincinnatiensis (−) (foodbornediarrhea) Aeromonas veronii (−) (wound infection, diarrhea) Aeromonascaviae (−) (gastroenteritis) Legionella pneumophila (−) (Legionnaires'disease) Treponema denticola (−) (necrotizing gingivitis) Porphyromonasgingivalis (−) (adult periodontitis) Tannerella forsythia (−)(periodontitis) Actinobacillus (−) (juvenile periodontitis)actinomycetemcomitans Neisseria meningitides (−) (meningitides)Neisseria gonorrhoeae (−) (gonorrhea) Neisseria sicca (−) (pneumonia,endocarditis) Haemophilus influenzae (−) (pneumoniae, otitis)Haemophilus ducreyi (−) (chancroid) Pseudomonas aeruginosa (−) (lung andskin infections) Pseudomonas pseudoalcaligenes (−) (peritonitis)Photorhabdus asymbiotica (−) (lesions) Salmonella enterica (s.Paratyphi) (−) (paratyphoid fever) Salmonella enterica (s. Typhi) (−)(typhoid fever) Klebsiella pneumoniae (−) (pneumonia) Yersinia pestis(−) (plague) Yersinia pseudotuberculosis (−) (Far East scarlet-likefever) Chlamydophila pneumoniae (−) (bronchitis, pneumonia) Chlamydiatrachomatis (−) (trachoma, vaginitis) Simkania negevensis Z (−)(juvenile pneumoniae and acute bronchiolitis) Waddlia chondrophila (−)(human fetal death) Pasteurella multocida (−) (lesions) Serratiaproteamaculans (−) (pneumonia) Bacteroides fragilis (−) (peritonealinfections) Moraxella catarrhalis (−) (respiratory, middle ear, eye, CNSinfections)

Illustrative examples of Na⁺-NQR-containing pathogens are found amongbeta- and gamma-proteobacteria (Enterobacteriales, Vibrionalles,Pasteurellales, Aeromonadales, Pseudomonadales, Neisserales),Bacteroidetes and Chlamydiae (Chlamydiae may have received Na⁺-NQR byhorizontal gene transfer).

Gram-positive Clostridiae (Clostridium difficile and other pathogenicClostridiae such as C. perfringens (Gangrene, Food poisoning), C. tetani(Tetanus), C. botulinum) have an ancestral form of the Na⁺-NQR enzyme,termed RFN. RFN contains a subunit RfnD, which is homologous to the NqrB(targeted by korormicin), but a Gly140-Gly141 pair from NqrB is notconserved in RfnD.

For illustrative purposes a compound with a furanone ring and N-linkedsubstituted hydrocarbon chain as characterized by a compound of formulaI, II, III or IV has been chemically synthesized and tested forantibiotic activity in a first set of experimental examples. Thecompound is referred to as PEG-2 and its chemical structure is:

Demonstration of PEG-2 antibiotic activity was carried out in a modelassay of Chlamydia trachomatis infection of HeLa cells. PEG-2 activitywas also compared to korormicin and various homoserine lactones. Thisfirst set of experimental examples are for illustration purposes onlyand is not intended to be a limiting description.

Chlamydia trachomatis (C. trachomatis) propagation and treatment werecarried out as follows. C. trachomatis was propagated in HeLa cells. Thetiter of C. trachomatis was determined in cyclohexamide-treated HeLacells, and concentrations used were expressed as inclusion forming units(IFU) per mL.

PEG-2 was solubilized in DMSO in (stock concentration 50 mM). During thetreatment with PEG-2 subsequent dilutions were made and correspondingamount of DMSO was always added to controls.

Cell Toxicity Assay was performed as follows. HeLa, HEK293 and primaryVSM cells were seeded at 5×10³ cells/well in 96-well plates andincubated with Korormicin, PEG-2 or homoserine lactones in DMEMcontaining 1% fetal bovine serum (FBS; Invitrogen Corp.). After 48hours, the number of living cells was determined by a colorimetricenzyme assay, based on a cytoplasmic enzyme activity present in viablecells.

Assessment of the level of infection was determined as follows. HeLacells were seeded on glass coverslips in 24-well plates at 3×10⁴cells/well and inoculated with C. trachomatis and then treated withantibiotic (or other compounds of interest). After 48 hours, theinfected cells were fixed with 100% methanol and then incubated withanti-Chlamydia monoclonal antibody. Inclusion bodies were visualized bystaining with FITC-conjugated secondary antibody and DAPI ascounterstaining. The samples were examined by a fluorescence microscopy.Number of inclusions was calculated per 100 cells.

Assessment of antichlamydial activity of Korormicin (Scheme shown inFIG. 1) and PEG-2 (Scheme shown in FIG. 2) were performed as follows.Cells were infected with C. trachomatis and treated with differentconcentrations of antibiotic of choice. After 48 h of C. trachomatis wascollected (P1) and used to infect fresh cells. Subsequent collections ofC. trachomatis were used to obtain P2, P3, P4 and P5 stocks of C.trachomatis. Dilution of C. trachomatis used to infect cells aftertreatment with PEG-2 had to be minimized in order to visualizeinclusions. (See scheme shown in FIG. 2). Last passage collection of C.trachomatis (P5) of Korormicin treatment and all collected of passagesof C. trachomatis treated with PEG-2 were used to establish infectivityof treated Chlamydia (Scheme shown in FIG. 3).

A comparison of the korormicin and PEG-2 structures is shown in FIG. 4.PEG-2 is a molecule which is a derivative of korormicin with the epoxygroup removed.

In 1997, Yoshikawa et al. (Japanese Patent 3905604B2 published 18 Apr.2007) isolated korormicin from the marine bacterium Pseudoalteromonassp. F-420, and demonstrated an inhibitory activity of korormicin againstthe growth of gram-negative marine Vibrios. Korormicin is the best, mostspecific inhibitor currently available to inhibit the Na⁺-NQR.Korormicin is a specific, very potent inhibitor of quinone reduction byNa⁺-NQR with the inhibitor constant of 82 pM (while HQNO has a Ki of 300nM). Both HQNO and korormicin have a common binding site in the Na⁺-NQRcomplex (mutually exclusive inhibitors). This indicates a possibleaccessibility problem in that only traces of the added antibioticactually reach its target (Na⁺-NQR). Furthermore, koromicin has neverbeen developed for use in animals, more particularly mammals, and evenmore particularly humans. Thus, a first experimental goal was todetermine the efficacy of korormicin against an intracellular infectionof animal cells, for example human cells. A second experimental goal wasto design an alternative to koromicin, and compare toxicity and/orefficacy results in the context of an intracellular infection of animalcells, for example human cells.

FIGS. 5 and 6 show that korormicin is effective against C. trachomatisinfection of eukaryotic cells, more particularly human cells. However,these concentrations of korormicin in cell culture model are ˜10,000fold higher (micromolar range) than in preparations of isolated Na⁺-NQR(Ki of ˜80 pM). Thus, these results appear to confirm the hypothesizedaccessibility problem. Furthermore, FIG. 7 shows that korormicin istoxic to the primary cells at the same concentrations in which it showsan antibiotic action. The toxicity problem of korormicin with respect toan animal cell, more particularly a human cell, is a novel finding thatwas previously unrecognized and has never been documented in priorpublished literature. These data clearly demonstrate that nativekorormicin is not useful as an antibiotic for treatment in humans.

FIG. 8 shows that PEG-2 is non-toxic to eukaryotic cells, moreparticularly human cells. Furthermore, examination of HeLa and HEK293cell lines as well as primary Vascular Smooth Muscle cells have found notoxic effects at PEG-2 concentrations of up to 50 μM (not shown).

FIGS. 9 through 13 show that PEG-2 is effective as an antibiotic againstC. trachomatis infection of eukaryotic cells, more particularly humancells. Moreover, FIG. 14 shows that PEG-2 is almost 1,000 times moreeffective as an antichlamydial agent than native korormicin. Thus, PEG-2improves upon korormicin for both toxicity and efficacy for antibiotictreatment of microbial infections of human cells. In addition, PEG-2represents an excellent platform for further drug design.

The possibility that homoserine lactones may function as antibiotics ineukaryotes was also studied. Representative homoserine lactones areshown in FIG. 15. The results provided in Table 2 show that thehomoserine lactones tested are either highly toxic(N-3-oxo-dodecanoyl-L-Homoserine lactone) to human cells or ineffective(the other three homoserine lactones) as anti-chlamydial agents. Theseresults suggest that the furanone ring of PEG-2 may be a significantcontributor to the efficacy of PEG-2 antibiotic activity.

TABLE 2 Effects of homoserine lactones on HeLa cell viability andanti-Chlamydia trachomatis efficacy. Cytotoxicity of PEG-2(concentration in μM) Anti-Chlamydial effect of On HeLa cell line PEG-2(concentration in μM) Name Formula 12.5 25 50 100 12.5 25 50 100N-decanoyl-L- C₁₄H₂₅NO₃ no no no no no no no no Homoserine lactoneN-butyryl-L- C₈H₁₃NO₃ no no no no no no No no Homoserine lactoneN-3-oxo- C₁₆H₂₇NO₄ yes yes yes yes n/a n/a n/a n/a dodecanoyl-L-Homoserine lactone N-hexanoyl-L- C₁₀H₁₇NO₃ no no no no no no No noHomoserine lactone

The mechanism of action of PEG-2 was also investigated by monitoringintracellular acidification of infected cells treated with PEG-2. IfPEG-2 is working through the inhibition of Na⁺-NQR and a sodium motiveforce, then it would be expected to have an effect on intracellular Na⁺and H⁺ concentrations within infected cells. HeLa cells were infectedwith C trachomatis and intracellular acidification was monitored withfluorescent indicator dyes.

C. trachomatis infection of a eukaryotic cell in culture acidifies thecytoplasm of the host cell at the onset of infection. As shown in FIGS.16 and 17, PEG-2 can delay the initial C. trachomatis-inducedacidification by at least 2 hours and diminishes the followingacidification.

Both of these effects on intracellular H⁺ are consistent with theproposed action of energy metabolism identified for a pathogenicbacterial infection and PEG-2's molecular mechanism of action throughthe inhibition of Na⁺-NQR activity.

Further synthesis of PEG molecules, including a stereoisomer of PEG-2,have been achieved and tested experimentally to demonstrate antibioticactivity in a second set of experimental examples. In this second set ofexperimental examples PEG-2 is the non-stereospecific furanoneantibiotic described above in the first set of experimental examples,while PEG-2S is a corresponding stereospecific furanone antibiotic. Thefollowing second set of experimental examples are for illustrationpurposes only and are not intended to be a limiting description.

Assays of the growth of free-living bacteria. Effect of the syntheticNa⁺-NQR inhibitor, PEG-2, on growth of E. coli, L. lactis, and E.faecalis was assayed as follows. Overnight starter cultures were grownaerobically in standard tryptic soy broth (TSB, Difco) and used toinoculate 200 μl TSB medium in 96-deep-well plates (Whatman) at aninitial OD600 of 0.05. The obtained cultures were supplemented with 0.5,1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 μM PEG-2 (or pure DMSO in “zero”controls) and grown at 37° C. for 24 h with vigorous aeration. At 6, 18,and 24 h, growth was measured as OD600 by scanning the plates on aBio-Rad iMark microplate reader. For samples taken at 18 and 24 h,serial dilutions with the pre-warmed growth medium were prepared usingthe aliquots of bacterial cultures. The experiments were repeated atleast three times.

Evaluation of a PEG-2S against Clostridium difficile (C. difficile ATCC700057) was performed by Drop method on pre-reduced BAK plates accordingto Performance Standards for Antimicrobial Susceptibility Testing(Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria;Approved Standard—Eighth Edition; Volume 32 Number 5, 2012).

Assessment of antichlamydial properties of antibiotics. For MIC50(Minimal Concentration of the antibiotic that inhibits formation ofchlamydial incluions on 50%) determination HeLa cells were grown in24-well or 96 well plates overnight prior to chlamydial inoculation.Elementary bodies were applied to the cells in small volume of SPG (0.22M sucrose, 8.6 mM Na₂HPO₄, 3.8 mM KH₂PO₄, 5 mM glutamic acid, 0.2μm-filtered, pH 7.4) and after 2 hours of incubation unattachedelementary bodies were removed and infected cells were treated withdifferent concentration of antibiotics in Dulbecco's modified Eagle'smedium (DMEM, Invitrogen) with 5% fetal bovine serum (FBS, Invitrogen)in the presence of cycloheximide (1.0 μg/ml). After 42 h inclusions werevisualized by immunocytochemi stry.

To determent chlamydicidal effect of Korormicin (Scheme 1) and PEG-2(Scheme 2) Hela cells were infected with C. trachomatis and treated withdifferent concentrations of antibiotics. After 48 h of C. trachomatiswas collected (P1) and used to infect fresh cells. Subsequentcollections of C. trachomatis were used to obtain P2, P3, P4 and P5stocks of C. trachomatis. Dilution of C. trachomatis used to infectcells after treatment with PEG-2 had to be minimized in order tovisualize inclusions. (See Scheme 2 below). Last passage collection ofC. trachomatis (P5) of Korormicin treatment and all collected ofpassages of C. trachomatis treated with PEG-2 were used to establishinfectivity of treated C. trachomatis. Minimal chlamydicidalconcentration (MCC2) for Korormicin, PEG-2 and PEG-2S was calculatedafter 2 passages of infection with treatment and reflect inhibition ofinfection for more than 90%. In all experiments inclusions werevisualized by immunocytochemistry.

Immunocytochemistry. Cells were seeded on glass coverslips in 24-wellplates (2-4×10⁴ cells/well) or on glass-bottom 96 well plates (10⁴cells/well), and after overnight inoculated with C. trachomatis. After42 hours, the infected cells were fixed with 90% acetone (or 100%methanol in case of 96 well plates) and then incubated withanti-Chlamydia antibody (Thermo Fisher Scientific). Alexa Fluor488-conjugated anti-rabbit IgG (Molecular Probes) was used as secondaryantibody. DAPI staining solution (300 nM) was added to the coverslips toidentify nuclei. Inclusion bodies were visualized using a fluorescentinverted microscope (TE-2000s; Nikon) Cells and inclusions were countedusing Adobe Stock Photos CS3.Ink software.

Cell Proliferation Assay. Cells were seeded at 5×10³ cells/well in96-well plates and incubated with C. trachomatis in DMEM containing 1%fetal bovine serum (FBS; Invitrogen Corp.). After 48 hours, the numberof living cells was determined by a colorimetric enzyme assay (CellTiter96 Cell Proliferation Assay; Promega Corporation, Madison, Wis.) basedon a cytoplasmic enzyme activity present in viable cells. (Cory et al.Use of an aqueous soluble tetrazolium/formazan assay for cell growthassays in culture. Cancer Commun. 1991; 3:207-212). The absorbance of aformazan product in tissue culture media was measured at 500 nm using amicroplate reader.

Preparation of sub-bacterial membrane vesicles from V. cholerae cells.Membrane vesicles from V. cholerae strains were prepared as describedpreviously (Dibrov et al. Chemiosmotic mechanism of antimicrobialactivity of Ag⁺ in Vibrio cholerae. Antimicrob Agents Chemotherapy.2002; 46:2668-2670) with some modifications. Mid-log cells wereharvested by centrifugation, washed once and resuspended in Buffer Acontaining 100 mM KCl, 50 mM NaCl, 5 mM MgSO4, 20 mM HEPES-Tris, pH 8.0.The cells were disrupted by a passage through the French press (Aminco)at 16,000 psi. Unbroken cells and cell debris were removed by low-speedcentrifugation, and the vesicles were collected after centrifugation ofthe supernatant at 180,000 g for 90 min. The membrane pellet wassuspended in buffer A at 20-30 mg protein/mL, snap-frozen in liquidnitrogen and stored at minus 80 degrees C. until use. Protein content insub-bacterial vesicles was determined by the Bio-Rad DetergentCompatible Protein Assay Kit.

Na⁺-NQR activity assays in sub-bacterial vesicles. Membranes of V.cholerae contain two enzymes capable of NADH oxidation: Na⁺-NQR andNDH-2 (non-coupled NADH:ubiquinone oxidoreductase of type 2), but onlyNa⁺-NQR is able to oxidize dNADH (deamino-NADH or nicotinamidehypoxanthine dinucleotide). Activity of Na⁺-NQR in sub-bacterial V.cholerae vesicles was measured at 25 degrees C. as oxidation of dNADH(ε₃₄₀=6.22 mM⁻¹ cm⁻¹) by following the changes in its fluorescence at440 nm (excitation light λ=340 nm) using Shimadzu RF-1501spectrofluorometer. The assays were conducted in Buffer A supplementedwith 15 μM Na⁺-dNADH with constant stirring. The reaction was initiatedby the addition of vesicles (aliquots of 50 μg of protein). Calibrationassays confirmed that fluorescence at 440 nm, as a function of [dNADH]in the experimental buffer was linear up to 20 μM of added dNADH (notshown).

Measurements of intracellular pH and sodium in cell cultures. pHrodo™Green AM and CoroNa Green Sodium Indicator (both Molecular Probes,Invitrogen) were used to measure intracellular pH (pH_(i)) andintracellular sodium (Na⁺ _(i)) concentration. HEK293 cells were seededon 24 well plates and infected with C. trachomatis after overnightincubation in DMEM with 5% FBS. After 2 hours of incubation with C.trachomatis cells were treated with different concentrations of PEG-2Sand subjected to pH_(i) and Na⁺ _(i) measurement at different timepoints according to manufacturer's protocols. In all assessmentscalibration curves were performed prior to the experiments. Fluorescentinverted microscope (TE-2000s Nikon) was used to obtain images and meanintensity was quantified using NIS Elements imaging software (Nikon,Mississauga, ON) and mean intensity was quantified using NIS Elementsimaging software (Nikon, Mississauga, ON).

Statistical Analysis Data are presented as mean±SEM unless otherwisestated. Differences between treatment groups were assessed by one-wayanalysis of variance using the Student-Newman-Keuls method. Aprobability of P≤0.05 was considered statistically significant.

Chlamydial invasion perturbs ion homeostasis of infected cells. Changesin cytoplasmic pH and Na⁺ content caused by the C. trachomatis infectingthe HEK293 cell culture are shown in FIG. 18. Internal pH in uninfectedcontrol did not change in the course of observation (values of ˜7.75 at2 hr and at 6 hr time points were identical to the pH in uninfectedcells of this lineage at zero time (not shown)), while in infected cellsby 2 hrs of infection cytoplasmic pH droped to ˜6.6 and reached ˜6.4 by6 hrs (FIG. 18A). Significant acidification of the cytoplasm in cellsinfected by Chlamydiae was hypothesized because at the onset of theinfection, this parasite rapidly consumes two preferable energysubstrates, ATP and glucose.

It was further hypothesized that a relative acidification of thecytoplasm together with depletion of the host ATP pool caused by themetabolic activity of chlamydial reticulate bodies (RBs) would (i) slowdown the Na⁺ export by Na⁺/K⁺ ATPase and (ii) stimulate Na⁺ uptake viaNa⁺/H⁺ antiporter(s) residing in the host cell membrane, thus resultingin a rise of intracellular sodium. Parallel measurements of internal Na⁺in uninfected and infected HEK293 cells presented in FIG. 18B, indeedshow a gradual increase of cytoplasmic Na⁺ in chlamydia-infected cells(black bars) but not in the control uninfected cells (empty bars)delayed relative to the observed acidification.

Inhibitor of chlamydial Na⁺-NQR, korormicin, suppresses proliferation ofRBs but it is toxic to mammalian cells. Like other members of theChlamydia genus, C. trachomatis encodes Na⁺-dependent symporters for theaccumulation of a number of important substrates, including amino acidsand dicarboxylates. Therefore, maintenance of the transmembrane sodiumgradient becomes a prerequisite for the proliferation of C. trachomatis.Since Na⁺-NQR is the major primary Na⁺ pump in this bacterium, itsinhibition might have a strong anti-chlamydial effect. Korormicin, anantibiotic initially isolated from a marine bacterium, Pseudoalteromonassp., is known to be an Na⁺-NQR inhibitor and a bactericidal agent thatis effective against marine bacteria but not against terrestrialmicroorganisms (Yoshikawa et al. Korormicin, a novel antibioticspecifically active against marine gram-negative bacteria, produced by amarine bacterium. J Antibiot. 1997; 50:949-953). Attractive features ofkorormicin are (i) its high efficiency (K_(i)≈8×10⁻¹¹ M in preparationsof isolated enzyme) and (ii) specificity, as it apparently has no effecton Na⁺-independent NADH oxidoreductases.

Korormicin was evaluated in the first set of experimental examples abovefor potential antibiotic targeting chlamydial Na⁺-NQR by examining itseffects on the growth of the C. trachomatis in cell culture models. Ithas been found that korormicin is effective against C. trachomatis whenadded at a concentration of 10 to 20 μM (FIGS. 5 and 6). Fluorescentmicroscopy images clearly indicated decrease in number of viable C.trachomatis cells in cell cultures treated with korormicin (FIG. 5).Sharp decline of infectivity after five consecutive treatments withkorormicin was evident already at 10 μM of added antibiotic; at 20 μM,number of chlamydial inclusions was less than 2% of control (FIG. 6).Calculated MIC₅₀ for korormicin (after 1^(st) treatment) was close to3.00 mM. This concentration exceeds the effective inhibitoryconcentration measured for isolated Na⁺-NQR by approximately six ordersof magnitude. Such a significant discrepancy does not seem altogethersurprising given an essential hydrophobicity of korormicin molecule(FIG. 4) and a number of membrane barriers, which potentially canscavenge korormicin before it gets to its target in situ. One could alsoargue that the antibiotic could be metabolized to some extent bymammalian and/or chlamydial cells.

Unfortunately, korormicin also had a palpable cytotoxic effect onmammalian cells (FIG. 7). Proliferation of the primary culture ofvascular smooth muscle cells (VSMC) was used as a sensitive experimentalmodel of cell response to a potentially toxic insult. As shown in FIG.7, cells were sensitive even to nanomolar concentrations of addedantibiotic; korormicin at concentrations of 5 to 15 μM lowered the celltiter by ˜10%. Thus, although the experiments with korormicin confirmedthe crucial role of Na⁺-NQR in the metabolism and, therefore,proliferation and infectivity of C. trachomatis, this inhibitor cannotbe used as anti-chlamydial remedy due to its toxic effect and lowefficiency in the cell culture model of infection, which could beregarded as a reasonable 1^(st) approximation of infected tissue.

Design of synthetic Na⁺-NQR inhibitor, PEG-2. Korormicin and anotherNa⁺-NQR inhibitor, HQNO, bind to the integral membrane subunit NqrB ofthe Na⁺-NQR complex, which harbors the transmembrae Na⁺ translocationpathway. HQNO and korormicin are mutually exclusive inhibitors, andtheir binding sites on NqrB are presumably overlapping although notidentical. The chemical structure of koromicin is shown in FIG. 4. Thefour isolated chiral centers in korormicin molecule allows eightpossible stereoisomers, but only one of them, the (5S,3′R,9′S,10′R)isomer (shown in FIG. 4) is the natural antibiotic.

Mode of action of both HQNO and korormicin indicates that thequinone-like “heads” may be the active structural modules responsiblefor binding the NqrB and that the geometry of polar groups may determinepotency of a given inhibitor. Both structures also have a prolongedaliphatic “tail”, which could be important for both crossing membranebarriers and docking of inhibitors to its target (they could be bindingNqrB from within the lipid bilayer). In koromicin, the conjugated diene(4′C-7′C), followed by the epoxy group at 9′C-10′C could play a role ofa rigid “spacer” separating the biologically active “head” of korormicinfrom a chaotic “tail”. The epoxy group at 9′C-10′C was identified as apossible source of korormicin toxicity. It is known to react withamino-, hydroxyl and carboxyl groups as well as inorganic acids. Delayedand immediate epoxy dermatitis has been reported. Certain epoxycompounds have mutagenic potential (i.e., they are potentiallycarcinogenic).

Taking all these structural considerations into account, design of anon-toxic Na⁺-NQR inhibitor took the following approach: (i) Toeliminate the cytotoxic effect of natural korormicin, its epoxy group,as a possible source of toxicity, was removed from the structure. (ii)Further, the aliphatic “tail” of korormicin was shortened to 7 carbonatoms to potentially lessen the overall hydrophobicity of the moleculeand thus lower its effective anty-chlamydial concentration. Thisapproach yielded a compound, PEG-2 (FIG. 4), which has been shown to bea potent Na⁺-NQR inhibitor and can be used as a structural platform forthe further development of Na⁺-NQR inhibitors.

Pharmacological properties of PEG-2. Due to the presence of two chiralcenters in the molecule, four stereoisomeres of PEG-2 are possible (seeFIG. 4). Preparations of PEG-2 obtained in the course ofnon-stereospecific synthesis carried out by Enamine Ltd (Kiev, Ukraine)were used in the first set of experimental examples described above.According to the manufacturer's report, this enantiomeric mixturecontained no more than 10% of the presumably active isomer shown in FIG.4.

Fluorescent microscopy confirmed that already a single treatment withPEG-2 had drastic effect on infection of HeLa cells by C. trachomatis(FIG. 9). Chlamydial inclusions formed in the presence of PEG-2 werehollow (FIG. 9, right image) compared to the control non-treated cellculture (FIG. 9, left image). They were also noticeably smaller (FIG.10). Thus PEG-2 retained anti-chlamydial activity. Direct comparison tokorormicin showed that enantiomeric mixture of PEG-2 is considerablymore effective (FIG. 14): after 5 consecutive treatments with 15 μMPEG-2, number of inclusions was 0.01% of control, while at the sameconcentration of natural korormicin it was 4.8%. Therefore, in thismodel of infection, anti-chlamydial effect of a given batch of PEG-2exceeded that of korormicin by at least 2 orders of magnitude. PEG-2acts in a dosage-dependent manner. As FIG. 19 shows, while the 1^(st)treatment with 15 μM enantiomeric mixture of PEG-2 resulted in ˜5-folddecrease of infectivity (measured as the number of chlamydial inclusionsin the next passage), after 5 consecutive treatments the number ofdetectable inclusions was 10,000 times lower. PEG-2S was even moreeffective as anti-chlamydial agent than PEG-2 (Table 3): MIC50 forPEG-2S was 0.7 μM and MIC50 for PEG-2 was 10 μM. This trend continuedafter two consecutive treatments: MIC50₂ for PEG-2S was 0.25 μM versus 4μM for PEG-2 (Table 3).

TABLE 3 Chlamydicidal properties of natural antibiotic Korormicin andsynthetic Na⁺-NQR inhibitor PEG-2. Korormicin PEG-2^(a) PEG-2S^(e)MIC50₁ ^(b) 18 μM (8.28 μg/ml) 10 μM (3.63 μg/ml)  0.7 μM (0.25 μg/ml)MIC50₂ ^(c) 10 μM (4.6 μg/ml)  4 μM (1.45 μg/ml) 0.25 μM (0.09 μg/ml)MCC2^(d) ND 15 μM (5.45 μg/ml)  1.0 μM (0.36 μg/ml) ^(a)Enantiomericmixture, PEG-2, contained no more than 10% of the biologically activestereoisomer PEG-2S (according to the manufacturer's report);^(b)Minimal Inhibitory Concentration after the treatment; ^(c)MinimalInhibitory Concentration after the second treatment; ^(d)MinimalChlamydicidal Concentration of added antibiotic; ^(e)Pure stereoisomer

PEG-2 is non-toxic and highly selective antibacterial agent PEG-2 aswell as PEG-2S provide a benefit of low cytotoxicity to mammalian cells:in contrast to natural korormicin, no toxic effects of PEG-2 on primarycell cultures (VSMC) were detected up to 20 μM of added PEG-2 (FIG. 8;compare to FIG. 7). Thus the koromicin epoxide at 9′C-10′C (see FIG. 4)apparently is the major reason for the cytotoxicity of koromicin.

One of the most appealing features of natural korormicin is its highselectivity as an inhibitor. It exclusively inhibits Na⁺-NQR, and thisenzyme has no homologues in mammalian cells as well as in the majorityof benign bacterial microflora and free-living species. In this respect,narrowly targeted or selective inhibitors of Na⁺-NQR would be “cleanantibiotics” with no unwanted side effects and minimized potential toprovoke an uncontrolled spread of drug resistance via mutant selectionin multiple environmental species and following lateral gene transfer,as presently occurs with conventional antibiotics.

As FIG. 20 shows, PEG-2S added at 1.0 to 50 μM concentrations did notaffect the growth of Na⁺-NQR-negative representatives of the benigngastrointestinal microflora, Escherichia coli (upper panel),Enterococcus faecalis (middle panel), and Lactococcus lactis (lowerpanel). Susceptibility tests of the growth of pathogenic Clostridiumdifficile to PEG-2S by a standard paper disc method, which is routinelyused to test antimicrobial action of korormicin, yielded the sameresult: PEG-2S does not affect the growth of C. difficile ATCC 700057 atthe concentrations tested (0.5 μM-50 μM) (data not shown). Of note,gram-positive C. difficile possesses the ancestral form of Na⁺-NQR, RNF.The Gly140 residue conserved in NqrB subunits of all known Na⁺-NQRenzymes and implicated in binding of korormicin, is absent in ahomologous subunit of RNF. One can therefore conclude that PEG-2 andPEG-2S upheld the high selectivity of inhibitory action that ischaracteristic for korormicin.

Direct measurements of inhibition of Na⁺-NQR by PEG-2S. Activity ofNa⁺-NQR could be measured in a number of experimental setups. In thecontext of this work, an especially attractive model is the registrationof the Na⁺-NQR-mediated oxidation of NADH in sub-bacterial membranevesicles. This approach can monitor Na⁺-NQR activity directly in realtime and can test Na⁺-NQR inhibitors with the enzyme operating in aphysiologically relevant background (being placed in its native membraneand feeding the electrons taken from NADH to the respiratory chain) butwithout additional permeability barriers and influences from cytoplasmicmetabolism.

Na⁺-NQR from the dangerous human pathogen Vibrio cholerae is the mostextensively studied representative of the class. This enzyme wastherefore chosen to test inhibitory properties of PEG-2. For theseexperiments in sub-bacterial vesicles, purified active stereoisomer ofPEG-2, PEG-2S (structure shown in FIG. 4), produced by stereospecificsynthesis by Canam Bioresearch Inc (Winnipeg, Canada), was used.

In addition to Na⁺-NQR, membrane of V. cholerae contains anotherNADH-oxidizing enzyme, non-coupled NDH-2 (non-coupled NADH:ubiquinoneoxidoreductase of type 2). While Na⁺-NQR oxidizes both NADH and itsanalog dNADH with similar rates, NDH-2 cannot use dNADH as a substrate.Therefore, the dNADH-oxidase activity of membrane vesicles could be usedto monitor Na⁺-NQR selectively. Indeed, elimination of functionalNa⁺-NQR by the chromosomal nqr A-F deletion results in the inability ofmembrane vesicles isolated from the mutant V. cholerae strain to oxidizedNADH (FIG. 21A, trace (a)). In contrast, vesicles isolated from theisogenic Na⁺-NQR-positive strain did oxidize dNADH in a PEG-2S-sensitivemanner (FIG. 21A, trace (b)). Titration of the Na⁺-NQR activity withPEG-2S in this experimental model yielded MIC₅₀ of 1.76 nM (FIG. 21B).For comparison, IC₅₀ for HQNO measured in the same experimental modelwas 130 nM.

PEG-2S disrupts chlamydial infection in cell culture model. Directmonitoring of cytoplasmic pH in HEK293 cells infected with C.trachomatis confirmed expectations (based on results shown in FIG. 18A)of rapid and deep acidification during the initial phase of chlamydialinfection (FIG. 22A, black triangles). While the subtle transientchanges of pH during first ˜5 hrs of infection in uninfected cells(empty triangles) were not statistically significant, the addition ofPEG-2S at 1.0 μM (empty squares) interrupted the chlamydia-causedacidification within first 5 hours of infection, resulting in almostcomplete relaxation of internal pH at ˜7.6. At 2.5 μM, PEG-2S preventedthe chlamydia-induced acidification completely (FIG. 22A, blackcircles). Of note, observed acidification of the cytoplasm in infectedHEK293 cells persisted for a long (up to 24 hrs) time. This could be dueto the high glucose content in the experimental medium used.

The initial acidification of the host cytoplasm should activate residentNa⁺/H⁺ exchange machinery, resulting in a rise of intracellular [Na⁺].As FIG. 22A shows, internal pH of infected cells indeed drops fromhomeostatic level of 7.75 to 6.5 after ˜5 hr of infection (FIG. 22A,black triangles). Apparently, this alarming acidification activatedNHE-type Na⁺/H⁺ exchanger(s) operating in the plasmalemma of HEK293cells, as it is evident from the significant rise in cytoplasmic [Na⁺](FIG. 22B, black triangles). As expected based on results shown in FIG.18, sodium accumulation was delayed relative to the acidification(compare FIGS. 18A and B; FIGS. 22A and B, black triangles). Again, theintracellular sodium accumulation was sensitive to the low μMconcentrations of PEG-2S (FIG. 22B, empty squares and black circles).

Taken together, the data summarized in FIG. 18A,B and FIG. 22A,B supportthe idea about the manipulation of ion homeostasis of infected cell byinvading chlamydia and demonstrate the importance of chlamydial Na⁺-NQRfor the infectious process.

These data support the use of PEG-2 and PEG-2S as an antibiotic and as aplatform for the further design of drug derivatives for alternativeantibiotics. Several illustrative prospective drug designs, includingPEG-3 and PEG-4, are provided in FIG. 23 in alignment with PEG-2.

As is apparent from FIG. 23, both PEG-3 and PEG-4 have a “shielded”positive charge at the distal end of the aliphatic module of themolecule. It is expected that, compared to PEG-2, PEG-3 will be (a) moresoluble and (b) able to accumulate in chlamydial cytoplasm against itsconcentration gradient. The guanidinum derivative, PEG-4 will sharethese beneficial features with PEG-3. In addition, it may potentiallyinhibit (very gently) the NHE-1 antiporter in the infected cells andthus lower intracellular [Na⁺] and potentially function to furtherimpede the development of chlamydial infection.

Further drug derivatives designed and synthesized from the PEG-2platform and have been tested for solubility and antibiotic activity.Antibiotic activity is assessed using the Chlamydia infection of cellculture experimental model described above in both the first and secondset of experimental examples. Fluorescence microscopy results shown inFIG. 24 indicate that treatment with each of the derivatives reducesinclusion size in Chlamydia infected cells.

Table 4 shows the summarized results of the testing with PEG-2S includedto provide reference values. The derivatives PEG-6(Boc), PEG-10, PEG-11,PEG-14 all showed improved solubility compared to PEG-2S, withPEG-6(Boc) and PEG-10 providing particularly high solubility in DMSO.Antibiotic activity was also demonstrated for all of the derivativesshown in Table 4 with comparable IC50 values for anti-chlamydialefficacy.

TABLE 4 PEG series of antibiotics targeting chlamydial Na⁺-NQR NameStructure IC₅₀(Chl), μM^(a) Solubility^(b) PEG-2S

0.70 +/− PEG-6(Boc)

0.96 +++ PEG-10

0.67 +++ PEG-11

0.63 + PEG-14

1.20 + ^(a)Concentration required for the half-maximal inhibition of theproliferation of Chlamydia trachomatis in cell culture experimentalmodel. ^(b)Solubility in DMSO as assessed by a qualified observer. “+/−”insoluble at concentrations exceeding 25 mM; “+” solubilized at 50 mMcompletely after 20 min at room temperature; “++” solubilized at 50 mMafter 5 min at room temperature; “+++” perfectly soluble in DMSO(instant solubilization at 50 mM)

The results provided herein support several novel findings. For example,the results support a novel method of antibacterial attack throughNa⁺-NQR inhibition, and provide a novel demonstration of antibiotictreatment of bacterial infection of animal cells through Na⁺-NQRinhibition. It should be recognized that utility of PEG-2 and relatedcompounds encompassed by formulae I, II, III or W are not to be limitedby theory or mechanism regarding Na⁺-NQR inhibition.

Another example of a novel finding is that the previously discoverednatural antibiotic korormicin can be improved by significantly reducingeukaryotic toxicity through removal of the epoxy group from its chemicalstructure with the creation of PEG-2. This marked reduction of drugtoxicity through removal of an epoxy group is novel and not recognizedby previous literature. Another example of a novel finding is that PEG-2provides improved efficacy as an antichlamydial agent than koromicin.This increased efficacy is novel and unrecognized by previousliterature. Another example of a novel finding, is that the supportivedata demonstrates potential and reasonable prediction for further drugdesign modifications of PEG-2 leading to efficacious molecules toinhibit Na⁺-NQR in a wide variety of pathogenic bacteria.

Embodiments described herein are intended for illustrative purposeswithout any intended loss of generality. Still further variants,modifications and combinations thereof are contemplated and will berecognized by the person of skill in the art. Accordingly, the foregoingdetailed description is not intended to limit scope, applicability, orconfiguration of claimed subject matter.

What is claimed is:
 1. An antibiotic compound of formula (III):

or a salt or stereoisomer thereof; wherein R¹-R³ and R⁵-R¹⁰ areindependently selected from the group consisting of H, alkyl group,substituted alkyl group, halogen, OH, NH₂ and SH; R⁴ is H, alkyl groupor substituted alkyl group; X¹-X² are independently selected from thegroup consisting of ═O, ═S, NH, H, alkyl, halogen, OH, SH and NH₂; W isa saturated acyclic hydrocarbon chain of 1 to 15 carbon atoms; and Z isa neutral or positively charged organic group.
 2. The compound of claim1, wherein the compound has only two chiral centers 5S and 3′R.
 3. Thecompound of claim 1, wherein R⁴ is H and X¹-X² are independentlyselected from ═O or OH.
 4. The compound of claim 1, wherein Z is aneutral charged organic group.
 5. The compound of claim 1, wherein Z isa positively charged organic group.
 6. The compound of claim 1, whereinZ is a triphenylphosphine group, a guanidine group, an aminoperimidinegroup or an amiloride group.
 7. The compound of claim 1, wherein R¹-R¹⁰are independently selected from H or a C1-C3 alkyl group, X¹-X² areindependently selected from the group consisting of ═O, ═S, H, halogen,OH and SH.
 8. The compound of claim 7, wherein R⁴ is H.
 9. The compoundof claim 8, wherein Z is an organic group comprising a nitrogen orphosphorus atom.
 10. The compound of claim 1 selected from the groupconsisting of:


11. The compound of claim 10, wherein the compound has only two chiralcenters 5S and 3′R.
 12. A pharmaceutical composition comprising anantibiotic effective amount of the compound of claim 1 and apharmaceutically acceptable carrier.
 13. A pharmaceutical compositioncomprising an antibiotic effective amount of the compound of claim 10and a pharmaceutically acceptable carrier.
 14. A method of treating abacterial disease comprising administering an effective amount of thecompound of claim 1 to a subject in need thereof, wherein the bacterialdisease is caused by a gram-negative bacteria.
 15. The method of claim14, wherein the bacterial disease is selected from the group consistingof cholera, acute gastroenteritis, ulcers, gastrointestinal infection,wound infection, septicemia, foodborne diarrhea, diarrhea,gastroenteritis, Legionnaires' disease, necrotizing gingivitis, adultperiodontitis, periodontitis, juvenile periodontitis, meningitides,gonorrhea, pneumonia, endocarditis, otitis, chancroid, lung infections,skin infections, peritonitis, lesions, paratyphoid fever, typhoid fever,plague, Far East scarlet-like fever, bronchitis, trachoma, vaginitis,juvenile pneumonia, acute bronchiolitis, human fetal death, peritonealinfections, respiratory infection, middle ear infection, eye infection,and Central Nervous System infection.
 16. The method of claim 14,wherein the bacterial disease is caused by an infection of a bacteriaselected from the group consisting of Vibrio cholera, Vibrioparahaemolyticus, Vibrio vulnificus, Vibrio gastroenteritis, Vibriodamsel, Vibrio fluvialis, Vibrio furnissii, Vibrio harveyi, Vibriohollisae, Vibrio costicola, Vibrio mimicus, Vibrio cincinnatiensis,Aeromonas veronii, Aeromonas caviae, Legionella pneumophila, Treponemadenticola, Porphyromonas gingivalis, Tannerella forsythia,Actinobacillus actinomycetemcomitans, Neisseria meningitides, Neisseriagonorrhoeae, Neisseria sicca, Haemophilus influenza, Haemophilusducreyi, Pseudomonas aeruginosa, Pseudomonas pseudoalcaligenes,Photorhabdus asymbiotica, Salmonella enterica (s. Paratyphi), Salmonellaenterica (s. Typhi), Klebsiella pneumonia, Yersinia pestis, Yersiniapseudotuberculosis, Chlamydophila pneumonia, Chlamydia trachomatis,Simkania negevensis Z, Waddlia chondrophila, Pasteurella multocida,Serratia proteamaculans, Bacteroides fragilis, and Moraxellacatarrhalis.
 17. The method of claim 14, wherein the bacteria is aspecies from a proteobacteria order selected from the group consistingof Enterobacteriales, Vibrionalles, Pasteurellales, Aeromonadales,Pseudomonadales, and Neisserales.
 18. The method of claim 14, whereinthe bacteria is a Bacteroidetes.
 19. The method of claim 14, wherein thebacteria is a Chlamydiae.
 20. The method of claim 14, wherein thebacteria is selected from the group consisting of Chlamydia trachomatis,Simkania negevensis Z, Candidatus Protochlamydia amoebophila UWE25,Chlamydia muridarum Nigg', Treponema denticola, Treponema putida,Porphyromonas gingivalis, Tannerella forsythia, Actinobacillusactinomycetemcomitans, Legionella pneumophila, Neisseria meningitides,Neisseria gonorrhoeae, Klebsiella pneumonia, and Chlamydophilapneumonia.
 21. The method of claim 14, wherein the compound is anantibiotic that is therapeutically effective in an amount that isnon-toxic to mammalian cells.